Axially adjustable chromatography column

ABSTRACT

Described is a column for separation of substance mixtures with a liquid. A cylindrical tube has a closure at each end. At least one of the closures is a movable piston. The piston has a socket which is radially expandable by axial pressing of two interacting conical parts for sealing of the piston against the inner wall of the column.

The present invention relates to a column for separation of substancemixtures, preferably on a preparative scale, with a liquid medium.

Liquid chromatography is used for separation of mixtures of substances.Thereby, such mixture is introduced in a column filled with anadsorptive agent, whereupon a liquid, referred to as an eluent, isallowed to flow through the column. Separation is achieved by thecomponents of the mixture being retarded to a different degree by theadsorptive agent. Liquid chromatography is used for preparative *oranalytical purposes. For analytical purposes, the substance mixture isintroduced in the column in a liquid phase, whereupon an eluent isallowed to flow through the column and into a detector which records theamount of passing substance.

Preparative chromatography is carried out with the purpose of producingpure compounds, or isolating compounds from a mixture of substances. Theamount of substance which is introduced into the column for such purposeis one to several powers of ten greater than what is introduced into acolumn for analytical purposes.

In preparative chromatography, in cases where the substance mixture isdissolved in a suitable liquid, the mixture may be introduced into thecolumn via tubing connected to the end of the column. In other cases,the substance mixture is introduced in a solid phase. A standardprocedure is to adsorb the mixture, in a separate operation, on anappropriate adsorbent material, which is thereafter introduced into thecolumn on top of the adsorptive agent. The substance mixture maycomprise synthetic products or natural products, e.g. plant extracts.Traditionally, long columns are used in this context, the top end ofwhich is open. The column is packed with an adsorptive agent to acertain level, followed by the substance mixture. The uppermost part ofthe column is used as a reservoir for the eluent, which is allowed topass through the adsorptive agent with the aid of gravity. This type ofcolumn allows for continuous supply of eluent. When higher flow ratesare desired, than what can be achieved with a hydrostatic pressurealone, the eluent may be forced through the column by closing the topend of the column and applying gas pressure. The latter method, ascurrently applied, is referred to as "flash chromatography". Althoughthis is an inexpensive solution, the method has drawbacks. Glass columnsmay explode by too high pressures applied, with the risk of glasssplinter flying about, and further, the system has to be decompressed onfilling or exchange of eluent. Decompression can cause formation ofblisters whereby inhomogenities occur in the packing of the adsorptiveagent.

Forcing of the eluent through the column with a pump bids greatadvantages. Supply of eluent and change of composition of the eluent canbe done in a continuous manner on the suction side of the pump. Withmedium pressures, the compressibility of the liquid is negligible. Dueto this, the risk of glass splinter flying about on breakage is small inuse of columns which completely filled up with solid phase and liquid.Due to said fact, it is advantageous to supply the eluent to the columnthrough a hollow piston, which can be brought into close contact withthe adsorptive material, whereupon it is made to seal against the wallof the column.

This type of chromatography is sometimes referred to as medium pressurechromatography, which is carried out at a pressure up to a few tens ofbar, e.g. 4 bar (0.4 MPa). Several manufacturers supply columns whererequirements of sealing against the column walls are complied with. Inmany instances, columns are employed, designed as a cylinder ofrelatively thick glass (2-8 mm) wherein the length of the column may bemade adjustable by means of a piston in the column. Usually the inlet oroutlet of the column runs through the piston and piston shaft inflexible tubing which exits at the end of the piston shaft,alternatively a tube is provided having a connection for flexible tubingopening at the end thereof. Usually the piston is provided with a devicewhich prevents it from rotating during compression of the O-ring.

Numerous inconveniences occur with this type of design. If the O-ring iscompressed too strongly, the column may rupture. If the piston shaft ismade of plastic material, too hard tightening of the screw may cause thepiston shaft to break or the compression threads to be damaged. Further,a dead lumen occurs between the lowermost part of the piston and theO-ring. A problem with such a column is that, on use of aggressiveliquid media, certain organic solvents in particular, the O-ring may beaffected and cause leakage and contamination of the liquid medium. Asmost columns on the market are intended for biochemical separations, noparticular attention has been paid, in choice of material for theconstructive details, to resistance against solvents such as chloroform,methylene chloride and ethyl acetate, which are usually occurringeluents in organic chemistry. These solvents may cause the O-ring toswell, which may bring about rupture of the column or render the pistondifficult to remove. A column provided with the mentioned O-ring seal,where the piston shaft is made of plastic material has been supplied byAmicon Ltd. Upper Mill, Stonehouse, Gloustershire GL10 2BJ GB.

Pharmacia LKB Biotecnology, Sweden have supplied a column named SRColumn System, which, in the place of an O-ring employs a piston havinga conical outer surface, and a ring arranged around said piston having aconical inner surface, where the conical surfaces are pressed againsteach other with a similar mechanism as in the column having the O-ringseal, thus that the ring will seal against the column wall. In thiscolumn it appears that the conical surfaces have the same cone angle,and the piston is locked in the column from the outset. With thisconstruction, the ring has to be strongly tightened initially, to avoidleakage. Thereby, like in the column having the O-ring seal, a riskoccurs of rupture of the column wall. Columns of the previously knownkinds are further impaired with the problem of a dead volume around theperiphery of the upper end of the piston.

According to the present invention, these drawbacks are avoided in acolumn for separation of substance mixtures with a liquid medium,comprising a cylindrical tube with a separation space therein having aclosure at each end, whereby the separation space has an inlet and anoutlet, respectively, through a channel in each end thereof, whereby atleast one of the closures is a movable piston trough which one of saidchannels runs and together with a channel in a piston shaft connected tothe piston makes up the inlet or the outlet, whereby said piston has asocket which is radially expandable by axial pressing of two interactingconical parts, for sealing of the piston against the inner wall of thecolumn. The invention is characterized in, that the piston comprises anexpandable socket abutting, in its resting position, against the innercylindrical surface of the column, and having a conical opening,widening towards the separation space with a certain cone angle, thatthe piston further comprises an inner part having an outer surfaceconically tapering in the direction away from the separation space andabutting with its periphery against the periphery of the conicalopening, that said inner part has a cone angle greater than the coneangle of the expandable socket, which inner part can be pressed againstthe expandable collar, by tightening means operable from the outside ofthe column, to cause a first expansion thereof, and that the channelledpiston shaft is lockable against the column by a locking means having acertain resilience in the longitudinal direction of the column, and thatsaid resilience is arranged thus that it allows for pressing said innerpart back under the action of the pressure of a liquid in the separationspace, to achieve a further expansion of the expandable socket, tosealing against the column wall.

By "cone angle" is intended the top angle between the generatrices ofthe imagined full conical surfaces in which the respective conicalsurface is comprised, in an unloaded state. The angle of the respectiveconical surface to the column wall is half the cone angle.

The expandable socket can be made with a bottom, and/or a washer orsimilar device may be placed between the expandable part and the pistonshaft. The inner part of the piston is designed in such way that, whenpressed against the expandable socket, it is not locked against thebottom thereof , the washer or the end of the piston shaft,respectively.

While the tightening means, operable from the outside, can consist of ascrew means arranged at the outer end of the piston shaft, as describedabove as known, in a preferred embodiment of the invention, thetightening means, operable from the outside is a thread means arrangedbetween the inner part of the piston and the piston shaft, which isoperable by turning the piston shaft.

In a further preferred embodiment of the invention, the locking meanshaving a certain resilience is a fitting threadable onto the column oran equivalent holding means, such as a snap device, holding a lid havinga built-in elasticity, which lid secures the piston shaft via securingmeans. In the case where the channel in the piston shaft is a tubingconnected to the channel of the piston, it is preferred that the tubingoutside the column runs through an opening arranged in the side of thepiston shaft. In all columns on the market the inlet and outlet tubingexits concentrically with the piston shaft, i.e. trough the outermostend of the piston shaft, or is connected to the outer end of the pistonshaft tube. This is disadvantageous, since the tubing will be folded anddamaged if the column is lowered against a support surface duringpacking of the column. In the present invention, the inlet or outlettubings are run out at the side of the piston shaft tube through a holeintended therefor, close to the outer end of the piston shaft tube. Bythis arrangement, the column may be supported on the piston shaft tubeon packing of the column, without the tubing being damaged. With sucharrangement, further, arrangement of a rotation device at the lower endof the piston shaft is facilitated.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is more closely described with reference to the encloseddrawing, showing a column according to an embodiment of the invention,partly in side view, partly in section (FIG. 1A) and a partialenlargement (FIG. 1B).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The column shown in the drawing comprises a column tube made of glass,having external threads at one end thereof and similar threads, notshown, at the other end of the column tube. The threads each engage athreaded cap 3,3'. An opening is shown in the planar end of the cap 3.In the column tube, a piston 5 and a similar piston 5' are movable. Thepistons together delimit a separation space 6, the volume of which isvariable by displacement of either or both pistons. In the piston 5 achannel 7 opens, which may be an outlet or an inlet, referred to here asan outlet channel. Said channel continues via a plug 8 in which a tubing9 is attached, which is a continuing portion of the outlet channel. Asimilar inlet channel, not shown, in the piston 5' is connected to atubing 9'. Tubing 9, 9' runs through a piston shaft 10, 10', and exitsthrough a hole 11, 11' arranged in the side of the piston shaft.

The piston 5 comprises an expandable part 12, made of elastic materialsuch as polytetrafluorethene. Part 12 comprises a collar 13, theperiphery of which abuts against the inner surface of the column tube.Collar 13 has an inner surface 13a conically opening with a certain coneangle, e.g. 15° in a column having an inner diameter of 15 mm, and 34°in a column having an inner diameter of 30 mm, and a bottom part 14integrated with the collar. Via a washer 15 the expandable part 12 issupported against the end of the piston shaft 10 directed towards theseparation space. An inner part 16 of the piston 5 has a conical outersurface 17, tapering conically in the direction away from the separationspace, said conical outer surface having a greater cone angle, e.g. 30°in a column having the diameter 15 mm and 50° in a column having thediameter 30 mm. The inner part 16 is attached via a threaded central tap16a engaging an inner thread in the piston shaft. When the inner part 16is forced into the expandable part 12, the conical surfaces 13a and 17are brought into contact with each other, whereby part 12 is pressedagainst the column wall.

A slotted disk 18 has a conical central part 19, which has inner threadswhich by radial compression of the disk can be brought into engagementwith threads 20 on the exterior of the piston shaft, by the conical partbeing pressed into a central opening in an elastically resilient disk21, the periphery of which is supported against a flange 22 left by theopening 4 in the end of the cap 3. A optional knurled end 23, 23', whichcan be replaced by a through-going turning pin, is shown arranged on theend of the piston shaft 10, 10'.

A manner of using the column shown as an example is described in thefollowing.

The piston 5, or 5' in the alternative, is adjusted to a desiredposition with the inner part 16 loosely abutting the collar 13. The cap3 is screwed onto the threads 2, whereby the resilient disk 21 pressesthe central part 19 of the slotted disk into engagement of the threads20. A first dilation of the collar 13 is brought about by turning thepiston shaft 10, whereby the friction against the inner wall of thecolumn tube 1 prevents parts 12 and 16 of the piston from rotating, atleast to a maximal torque, at which rupture of the column is preventedby the piston starting to rotate. The maximal torque can be influencedby selection of material in the parts of the piston and selection of thearea by which the collar 13 abuts the inner wall of the column. Theseparation space 2 is filled with an adsorptive agent and the second ofpistons 5,5' is adjusted into a desired position and is locked in asimilar manner as the first one. The mixture that is to be separated isfilled via the inlet channel 9' (9 in the alternative). Eluent isintroduced under an moderate overpressure through the inlet channel, andthe liquid which flows out through the outlet channel is collected infractions. The eluent exerts a pressure on the upper side of the innerpart of the column. By the resilient disk allowing the piston shaft tomove outwards, the inner part 16 of the piston is pressed into theexpandable part 12, which expands. It is important that there is an openspace 12a between the bottom part and the inner part of the piston, thusthat expansion can be achieved in the two expansion phases.

While the column tube is normally made of glass, the expandable socketof the piston is preferably made of polytetrafluorethene or a similarpolymer material inert against occurring solvents. The inner part of thepiston, as well as the piston shaft and the supporting disk, whenoccurring, can be made of highly alloyed corrosion resistant steel, butstrong polymer materials known for such purposes can also be employed,whereby care should be taken in order that the parts of the inner parttouched by the liquid medium should be as inert against the liquidmedium as is the expandable socket.

I claim:
 1. A column for separation of substance mixtures with a liquidmedium, comprising a cylindrical tube (1) with a separation space (6)therein having a closure at each end, whereby the separation space hasan inlet and an outlet, respectively, through a channel (7,9) in eachend thereof, whereby at least one of the closures is a movable piston(5) through which one of said channels runs and together with a channelin a piston shaft (10) connected to the piston makes up the inlet or theoutlet, whereby said piston has a socket which is radially expandable byaxial pressing of two interacting conical parts, for sealing of thepiston against the inner wall of the column, wherein the piston (5)comprises an expandable socket (12) abutting, in its resting position,against the inner cylindrical surface of the column, and having aconical opening, widening towards the separation space with a certaincone angle, that the piston further comprises an inner part (16) havingan outer surface conically tapering in the direction away from theseparation space and abutting with its periphery against the peripheryof the conical opening, that said inner part has a cone angle greaterthan the cone angle of the expandable socket, which inner part (16) canbe pressed against the expandable collar, by tightening means operablefrom the outside of the column, to cause a first expansion thereof, andthat the channelled piston shaft (10), arranged to the piston, islockable against the column by a locking means (18) having a certainresilience in the longitudinal direction of the column, and that saidresilience is arranged thus that it allows for pressing said inner part(16) back under the action of the pressure of a liquid in the separationspace, to achieve a further expansion of the expandable socket (12), tosealing against the column wall.
 2. A column according to claim 1,wherein the tightening means, operable from the outside, is a threadmeans (16a) arranged between the inner part of the piston and the pistonshaft, which is operated by turning the piston shaft (10).
 3. A columnaccording to claim 1 or 2, wherein the locking means having a certainresilience is a fitting threadable onto the column and holding a lid(18) having a built-in elasticity, which lid secures the piston shaftvia securing means (19).
 4. A column according to claim 1 or 2, whereinthe channel in the piston shaft is a tubing connected to the channel ofthe piston, the tubing outside the column runs through an opening (11)arranged in the side of the piston shaft.